The human NSCLC cell lines A549, H1299 and H1975 and the normal human lung epithelial cell lines BEAS-2B were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. RPMI-1640 medium and PBS were purchased from Corning, Inc. and FBS was purchased from PAN-Biotech GmbH (cat. no. P30-3302). All cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS at 37°C in a humidified atmosphere with 5% CO₂. SchA, with a purity of >98%, was purchased from Chengdu Must Bio-Technology Co., Ltd. (cat. no. 19092908). SchA was dissolved DMSO at a concentration of 50 mM for subsequent experiments.

The A549, H1299, H1975 and BEAS-2B cell lines were seeded into a 96-well plate at a density of 3,000 cells per well and incubated at 37°C overnight. For concentration dependence experiments, different concentrations of SchA (0, 25, 50, 75 and 100 µM) or an equal volume of DMSO were added to the cell culture medium to treat the cells for 24 h at 37°C. For time dependence experiments, the cells were treated with 50 µM SchA for 0, 12, 24, 36 and 48 h at 37°C. A Cell Titer-Glo Luminescence Viability Assay kit (cat. no. G7571; Promega Corporation) was used to detect cell viability according to the manufacturer's protocol. Each concentration and time dependence assay was repeated three times. The IC₅₀ and its 95% confidence interval of SchA was evaluated using best-fit dose-response inhibition curves with GraphPad Prism v7.0 Software (GraphPad Software, Inc.).

The effect of SchA on cell colony formation was evaluated using colony formation assays. Single-cell suspensions of A549 and H1975 were seeded into a six-well plates at a density of 800 cells/well. After the cells adhered, SchA at concentrations of 0, 25 and 50 µM was added into the culture medium. The cells were placed in an incubator and cultured for 10-14 days at 37°C until a single cell clone contained ~50 cells. The colonies were then fixed with 4% paraformaldehyde for 1 h at room temperature and stained with 0.2% crystal violet for 20 min (Sigma-Aldrich; Merck KGaA) at room temperature. The number of colonies visible to the naked eye (Each colony contains >50 cells; ×1 magnification) were counted using ImageJ v2 software (ImageJ; National Institutes of Health).

The cells were trypsinized with trypsin and centrifuged at 300 × g for 3 min at room temperature, before being re-suspended in 1 ml pre-chilled 70% ethanol. After fixation overnight at -20°C, the cells were re-suspended and stained with 500 µl PI/RNase staining buffer (cat. no. 550825; BD Biosciences) at a density of 1×106 cells/ml at room temperature for 30 min. The cells were then detected using a CytoFLEX analysis flow cytometer (Beckman Coulter, Inc.) and the FL2-A: B610-ECD channel. The results were analyzed using the FlowJo v10 software (FlowJo LLC).

After the A549 and H1975 cells were treated with SchA (0, 25 and 50 µM) for 24 h at 37°C, the cells were trypsinized without EDTA (cat. no. CR27250; Guangzhou Ruisen Biotechnology Co., Ltd.). After washing twice with pre-chilled PBS, the cells were re-suspended in 1X buffer provided in the FITC Annexin V Apoptosis Detection kit (cat. no. 556547; BD Biosciences) to 1×10⁶ cells/ml. Next, 100 µl cell suspension was added to a flow cytometry tube and 5 µl Annexin V-FITC and 5 µl PI staining solution (cat. no. 556547; BD Biosciences) was added. The samples were incubated at room temperature for 15 min in the dark, before 400 µl 1X buffer provided in the kit was added. A CytoFLEX analysis flow cytometer (Beckman Couler, Inc.) and the FL1-A: B525-FITC and FL4-A: Y585-PE channels were used to measure cell apoptosis, where the results were analyzed using the FlowJo v10 software (FlowJo LLC).

The A549 and H1975 cells were also treated with 5 mM 3-MA for 4 h at 37°C and then treated with 50 µM SchA for 24 h at 37°C. The apoptosis of the cells was measured by the FITC Annexin V Apoptosis Detection kit (cat. no. 556547; BD Biosciences) and a CytoFLEX analysis flow cytometer (Beckman Coulter, Inc.). The results of apoptosis were also analyzed using the FlowJo v10 software (FlowJo LLC).

A DNeasy Blood & Tissue kit (cat. no. 69504; Qiagen GmbH) was used to extract DNA from the cells based on the manufacturer's protocols. The DNA sample was mixed with 10X loading buffer (cat. no. 317051; Vazyme Biotech Co., Ltd.) at a ratio of 9:1 before being separated in 1% agarose gels with the 0.01% Ultra GelRed nucleic acid stain (cat. no. GR501-01; Vazyme Biotech Co., Ltd.). A 200-bp DNA Ladder (cat. no. 3423A; Takara Bio, Inc.) was used to calculate the size of the DNA molecules. The FluorChem FC3 System (ProteinSimple) was used for gel imaging and the AlphaView v1.0 software (ProteinSimple) was used to analyze the density of the DNA bands.

The single-cell suspensions of A549 and H1975 were seeded into six-well plates at a density of 2×10⁵ cells/well. After the cells were treated with SchA (0, 25 and 50 µM) for 24 h at 37°C, the cells were washed twice with PBS and incubated with Hoechst 33342 (1 µg/ml; cat. no. C1029; Beyotime Institute of Biotechnology) for 15 min at room temperature, followed by incubation with PI (5 µg/ml) for another 15 min at room temperature. Subsequently, the cells were washed three times with PBS and the blue and red fluorescence emitted by the cells was observed. Images were captured using a fluorescence microscope (Leica Microsystems GmbH) at ×200 magnification. Image-Pro Plus v6.0 software (Media Cybernetics, Inc.) was used to merge the monochromatic fluorescence images.

A MMP Detection kit (MitoScreen; cat. no. 551302; BD Biosciences) was used to measure the level of damage to the MMP in the cell. The JC-1 dye was diluted into a JC-1 working solution according to the manufacturer's protocol. The cells were re-suspended with the JC-1 working solution at a density of 1×10⁶ cells/ml and incubated at 37°C for 15 min. After staining, the cells were centrifuged at 240 × g for 3 min and washed twice with 1X buffer provided in the kit. The cells were re-suspended in 500 µl 1X buffer provided in the kit and detected using a CytoFLEX analysis flow cytometer (Beckman Coulter, Inc.), with the FL1-A: B525-FITC and FL4-A: Y585-PE channels. Red JC-1 dye fluorescence represents live cells with intact MMP whereas green JC-1 monomer fluorescence represents apoptotic or dead cells with impaired MMP. The ratio of red (JC-1 aggregates) to green (JC-1 monomers) fluorescence intensity was used to assess MMP using the FlowJo v10 software (BD Biosciences).

The single-cell suspensions of A549 and H1975 were seeded into a six-well plates at a density of 2×10⁵ cells/well. The cells were treated with SchA (0, 25 and 50 µM) for 24 h at 37°C, then ROS probe DCFH-DA (cat. no. 287810; Sigma-Aldrich; Merck KGaA) was added to the cell culture medium at 1:1,000 before the cells were incubated at 37°C for 20 min and washed twice with PBS. Intracellular ROS levels were detected using a fluorescence microscope at ×200 magnification (Leica Microsystems GmbH).

After the cells were treated with SchA (0, 25 and 50 µM) for 24 h at 37°C, the cells were trypsinized to form a single cell suspension with trypsin and re-suspended. The cells were re-suspended in medium containing 0.1% DCFH-DA at a density of 1×10⁶ cells/ml and incubated at 37°C for 20 min. They were then washed twice with PBS and re-suspended with 500 µl PBS after centrifugation at 240 × g for 3 min at 37°C. The FL1-A: B525-FITC channel in a CytoFLEX analysis flow cytometer (Beckman Coulter, Inc.) was used to detect the level of intracellular ROS. The flow cytometry data was analyzed using the FlowJo v10 software (FlowJo LLC).

MDC was purchased from Sigma-Aldrich (cat. no. 10121-91-2; Sigma-Aldrich; Merck KGaA). The single-cell suspensions of A549 and H1975 were seeded into a six-well plates at a density of 2×10⁵ cells/well. After treatment with SchA (0, 25, 50 µM) for 24 h at 37°C, the cells were washed twice with PBS and incubated with MDC (final concentration, 0.05 mM) and Hoechst 33342 (final concentration, 1 µg/ml) for 15 min at room temperature in the dark. After washing the cells three times with PBS, the green and blue fluorescence emitted by the cells, was observed. Images were captured using a fluorescence microscope at ×200 magnification (Leica Microsystems GmbH). Image-Pro Plus v6.0 software (Media Cybernetics, Inc.) was used to merge the monochrome fluorescent images. Autophagy inhibitor 3-methyladenine (3-MA) was purchased from Sigma-Aldrich (cat. no. M9281; Sigma-Aldrich; Merck KGaA).

The ATP levels were assessed by ATP Detection Assay kit (cat. no. 700410, Cayman Chemical Company) according to the manufacturer's protocols. The single-cell suspensions of A549 and H1975 were seeded into a six-well plates at a density of 2×10⁵ cells/well. After the cells were treated with SchA (0, 25 and 50 µM) for 24 h at 37°C, the cell culture medium was first discarded and the cells were washed with pre-chilled PBS before 1 ml 1X sample buffer was added to each well (6-well plate) to lyse the cells. D-luciferin and luciferase were added to the ATP detection buffer to prepare the reaction mixture. The detection holes on the black 96-well plates and three duplicate wells for each sample were organized. A total of 100 µl freshly prepared reaction mixture and 10 µl cell lysate were added to each well, mixed and incubated for 15 min in the dark at room temperature. A multifunctional microplate reader was used to measure the fluorescence value in each well. GraphPad Prism v7.0 software (GraphPad Software, Inc.) was used to generate a histogram to show the fluorescence values.

The cells were lysed using the RIPA Lysis Buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) containing the Protease Inhibitor Cocktail (cat. no. 05892791001; Roche Diagnostics GmbH) and Phosphatase Inhibitor Cocktail (cat. no. 524629; EMD Millipore). The BCA Protein Assay kit (cat. no. PC0020; Beijing Solarbio Science & Technology Co., Ltd.) was used to determine protein concentration. Loading buffer (5X) was then added to the protein lysate at a ratio of 1:4. A total of 30 µg protein sample per line was separated using 12% SDS-PAGE and then transferred onto PVDF membranes (EMD Millipore). The membranes were blocked with 5% BSA (cat. no. 97061-422; VWR International) in TBS-0.1% Tween-20 (TBST) before being incubated with primary antibodies on a shaker overnight at 4°C. The p53 (cat. no. 2527), p21 (cat. no. 2947), cyclin D1 (cat. no. 2922), CDK4 (cat. no. 12790), CDK6 (cat. no. 13331), cyclin E1 (cat. no. 20808), cyclin E2 (cat. no. 4132), CDK2 (cat. no. 2546), capspase-3 (cat. no. 14220), poly (ADP-ribose) polymerase (PARP; cat. no. 9542), Bcl2-like 11 (Bim; cat. no. 2933), Bcl2 (cat. no. 4223), Bax (cat. no. 5023), caspase-9 (cat. no. 9502), LC3 (cat. no. 4108), p62 (cat. no. 8025), phosphorylated (p-) AMPKα-T172 (cat. no. 2535) and AMPKα (cat. no. 5831) antibodies were purchased from Cell Signaling Technology, Inc. The primary antibodies against SOX4 (cat. no. DF2610; Affinity Biosciences) and β-actin (cat. no. A1978; Sigma-Aldrich; Merck KGaA) were also used. All primary antibodies were diluted in primary antibody dilution buffer (cat. no. P0256; Beyotime Institute of Biotechnology) at a ratio of 1:1,000. The membrane were washed three times with TBST and then incubated with HRP-conjugated anti-rabbit (cat. no. 7074; Cell Signaling Technology, Inc.) or anti-mouse (cat. no. 7076; Cell Signaling Technology, Inc.) secondary antibodies at room temperature for 1 h. After washing the membrane three times with TBST, an ECL kit chemiluminescent substrate (cat. no. BL523B; Biosharp Life Sciences) was used to visualize the protein bands.

Total RNA was extracted using a RNA Quick Purification kit (cat. no. ES-RN001; Shanghai YiShan Biotechnology Co., Ltd.) and reverse transcribed into single-strand cDNA using a HiScript II Q RT SuperMix kit (cat. no. R223; Vazyme Biotechnology Co., Ltd.) at 50°C for 15 min, followed by 85°C for 5 sec. Quantitative PCR (qPCR) was performed using the PowerUp™ SYBR™-Green Master Mix (cat. no. A25742; Thermo Fisher Co., Ltd.) and an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Inc.). The thermocycling conditions of qPCR were set as follows: 95°C for 1 min, followed by 95°C for 10 sec, 60°C for 20 sec and 72°C for 30 sec for 40 cycles. Relative expression levels were calculated by the 2^−ΔΔCq method normalized to the internal reference gene β-actin (47).

Reverse transcription (RT)-semiquantitative PCR (RT-PCR) was conducted using 2X Taq Master mix (cat. no. P112; Vazyme Biotech Co., Ltd.) and a Bio-Rad T100 thermal cycler (Bio-Rad Laboratories, Inc.). The thermocycling conditions of RT-PCR were set as follows: 95°C for 3 min, followed by 95°C for 15 sec, 60°C for 15 sec and 72°C for 60 sec for 25 cycles. The primer sequences were as follows: Bim forward, 5′-TAAGTTCTGA GTGTGACCGAGA-3′ and reverse, 5′-GCTCTGTCTGTA GGGAGGTAGG-3′; Bim (BimEL, BimL and BimS), which contained exons 2 and 5 (48), forward, 5′-ATGGCAAAGC AACCTTCTGA-3′ and reverse, 5′-TCAATGCATTCTCCACA CCA-3′ and β-actin forward, 5′-CATGTACGTTGCTATCCA GGC-3′ and reverse, 5′-CTCCTTAATGTCACGCACGAT-3′. A total of 10 µg DNA per lane was separated using 1% agarose gel and stained with 0.01% Ultra GelRed nucleic acid stain (cat. no. GR501-01; Vazyme Biotech Co., Ltd.).

All experiments were repeated 3 times for each assay and the results were presented as the mean ± SD. One-way ANOVA was used to evaluate the quantitative data for statistical significance, then Dunnett's test was used for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

The 2D and 3D structures of SchA (PubChem compound ID, 43595; C₂₄H₃₂O₆; molecular weight, 416.5 g/mol) are shown in Fig. 1A and B. SchA belongs to the dibenzocyclooctadiene family of lignans (49). Its main feature is that the biaryl units are connected via aliphatic chains to form an eight-membered ring carbon skeleton (50).

The CellTiter-Glo assay was used to measure the viability of cells treated with different concentrations of SchA (0, 25, 50, 75 and 100 µM) for 24 h. The results showed that SchA exerted inhibitory effects on the viability of A549, H1975, H1299 and BEAS-2B cells in a concentration-dependent manner (Fig. 1C). The IC₅₀ and its 95% confidence interval (CI) were used to evaluate the ability of SchA to inhibit cell viability. The IC₅₀ and 95% CI in the A549, H1299, H1975 and BEAS-2B cell lines were 61.09 (44.47-177.0), 101.5 (65.45-529.2), 39.99 (33.48-59.28) and 49.45 (44.84-57.16) µM, respectively. The A549 and H1975 cell lines appeared to be more sensitive to SchA, whilst the H1299 cell line was more resistant. As the treatment time with 50 µM SchA was prolonged (0, 12, 24, 36 and 48 h), the viability of the A549 and H1975 cell lines was also markedly reduced (Fig. 1D). SchA (50 µM) exerted a potent inhibitory effect on the viability of A549 and H1975 cells 24 h after treatment (Fig. 1C and D).

Colony formation assay was used to detect the effect of SchA on cell proliferation and colony formation. The results showed that SchA could significantly inhibit the colony formation abilities of A549 and H1975 cells, where this inhibitory effect was concentration-dependent (Fig. 1E and F).

The biological process that is most closely associated with cell proliferation is cell mitosis. Therefore, to investigate the effect of SchA on mitosis, the cell cycle was evaluated using flow cytometry after PI staining. After treatment of A549 and H1975 cells with increasing concentrations of SchA (0, 10, 20, 30 and 50 µM) for 24 h, cell cycle progression was assessed. As shown in Fig. 2A and B, after the cells were treated with lower concentrations of SchA (10-20 µM), the percentage of cells in the G₀/G₁ phase was significantly increased. However, after the cells were treated with higher concentrations of SchA (20-50 µM), the percentage of cells in the G₀/G₁ phase was gradually decreased (Fig. 2A and B). Although the percentage of cells in S phase and G₂/M phase gradually increased, no statistical significance could be found. In addition, as the concentration of SchA increased, the proportion of cells in the sub-G₁ phase was also significantly increased. This suggest that lower concentrations of SchA (10-20 µM) can induce G₁/S-phase arrest, whilst higher concentrations of SchA (20-50 µM) can induce apoptosis.

Western blot analysis was subsequently used to measure changes in the expression levels of cell cycle-related proteins. As the concentration of SchA was increased (0, 10, 20, 30 and 50 µM), the protein expression levels of p53, cyclin E2, CDK2 and SOX4 were increased, whilst the protein expression levels of cyclin D1, CDK4, CDK6 and cyclin E1 were decreased (Fig. 2C and D). The protein expression levels of p21 were increased initially, peaking at 10 µM SchA before decreasing again at higher concentrations (Fig. 2C). After the cells were treated with 50 µM SchA for increasing periods time (0, 3, 6, 12 and 24 h), the protein expression levels of p53, cyclin E2, CDK2 and SOX4 were increased, whereas the protein expression levels of p21, cyclin D1, CDK4, CDK6 and cyclin E1 were decreased (Fig. 2D).

Sub-G₁ cells are hypodiploid cells with an incomplete genome (51). SchA was previously found to increase the percentage of sub-G₁ cells whilst also increasing the expression of the DNA damage-related proteins p53 and SOX4 (52). This suggests that DNA damage and apoptosis occurred in the cells following treatment with SchA. In the present study, the percentage of apoptotic cells was detected using flow cytometry and Annexin V-FITC/PI double-staining. The results indicated that the number of early and late apoptotic cells was significantly increased with increasing SchA concentrations (Fig. 3A and B). In addition, DNA electrophoresis was used to detect the effect of SchA on DNA integrity. It was found that SchA treatment caused DNA to break into a characteristic ladder-like pattern (Fig. 3C and D). To investigate the apoptosis-related morphological changes, the SchA-treated cells were stained with Hoechst 33342 and PI before being observed using fluorescence microscopy. The results showed that the cells treated with SchA were shrunken, with evidence of fragmentation and emitted irregular dense masses of blue fluorescence (Fig. 3E and F). In addition, PI red fluorescence was also increased by SchA treatment (Fig. 3E and F).

To verify the effects of SchA on apoptosis on the molecular level, western blot analysis was performed to measure changes in the protein expression levels of apoptosis-related proteins in cells treated with SchA. Following SchA treatment, caspase-3 and PARP in A549 and H1975 cells were cleaved, such that the expression levels of cleaved caspase-3 and cleaved PARP were markedly increased (Fig. 3G and H). These findings suggest that SchA induced apoptosis in A549 and H1975 cells.

Mitochondrial damage is one of the key features of apoptotic cells, where the loss of MMP is an essential manifestation of mitochondrial damage (53). To investigate the effect of SchA on mitochondrial physiology, JC-1 staining was used to detect the integrity of the MMP in A549 and H1975 cells after treatment with SchA. The red JC-1 aggregates represent intact MMP, whilst green JC-1 monomers represents damaged MMP. As the concentration of SchA increased (0, 25 and 50 µM), the number of cells emitting red fluorescence was decreased, whilst the number of cells emitting green fluorescence was increased (Fig. 4A and B). As a result, there was a significant decrease in the ratio of red (JC-1 aggregates)/green (JC-1 monomers) fluorescence (Fig. 4A and B), suggesting that SchA induced MMP damage in A549 and H1975 cells.

Mitochondrial damage leads to impaired oxidative phosphorylation and electron transport chain, which results in excessive ROS production and release from mitochondria (54). Excessive and abnormally distributed ROS can also affect mitochondrial function and reduce cell viability (54). To detect whether the ROS level was affected by SchA, DCFH-DA probes were used to evaluate intracellular ROS levels in the A549 and H1975 cells. The results of DCFH-DA staining were first detected using flow cytometry (Fig. 4C and D). As the concentration of SchA was increased, the number of cells emitting green fluorescence was also markedly and gradually increased, with the green fluorescence intensity strengthening progressively.

The results of DCFH-DA probe staining were also observed using fluorescence microscopy. As shown in Fig. 4E and F, after SchA treatment DCFH-DA green fluorescence in A549 and H1975 cells increased in intensity, which was widely distributed in the cytoplasm. These findings indicate that SchA increased the levels of intracellular ROS and disrupted the distribution of ROS in A549 and H1975 cells.

To verify if SchA induces cell apoptosis by activating the mitochondrial pathway, western blot analysis was used to detect the expression levels of mitochondrial apoptosis pathway-related proteins. As shown in Fig. 4G and H, after SchA treatment the expression levels of pro-apoptotic mitochondrial proteins, BimEL, BimL, BimS and Bax in A549 and H1975 cells were markedly increased. By contrast, the expression level of the anti-apoptotic protein Bcl-2 was decreased. In addition, the expression level of mitochondrial apoptosis biomarker, cleaved-caspase 9 (55), was markedly increased.

Bim is involved in sensing cellular stress and initiates the mitochondrial apoptosis pathway (56). Bim gene transcription typically increases after the cellular stress response and is expressed in the form of various alternative splice variants, including BimEL, BimL and BimS (48,57). Subsequently, it was found that the protein expression levels of BimEL, BimL and BimS in the cells were increased after SchA treatment. This may be due to reduced degradation or increased expression of the Bim protein. To detect the effect of SchA on the mRNA expression level of the Bim gene, RT-qPCR was used to detect the mRNA expression levels of Bim. As shown in Fig. 4I, SchA increased of the mRNA expression levels of total Bim in A549 and H1975 cells. In addition, the expression of the mRNA levels of BimEL, BimL and BimS in the A549 and H1975 cells were also measure through RT-PCR. It was found that the mRNA expression levels of BimEL, BimL and BimS were also increased after SchA treatment (Fig. 4J). These results suggest that SchA enhanced Bim gene transcription in A549 and H1975 cells.

To investigate the effect of SchA on autophagy in A549 and H1975 cells, they were treated with SchA and stained with MDC and Hoechst 33342. Results were then observed by fluorescence microscopy. In the control cells, Hoechst 33342 emitted weak and uniform blue fluorescence whereas MDC emitted uniform yellow/green fluorescence. In the SchA treated cells, Hoechst 33342 showed irregularly concentrated blue fluorescence whilst MDC showed dense green bodies in the cytoplasm (Fig. 5A and B). The blue fluorescence of Hoechst 33342 and the yellow/green fluorescence intensity of MDC were increased as the SchA concentration was increased (Fig. 5A and B).

In addition, a multifunctional microplate reader was used to detect the fluorescence intensity of the MDC-stained cells. Compared with that in the control cells, the green fluorescence intensity in A549 and H1975 cells treated with SchA was significantly increased (Fig. 5C and D).

To verify that SchA activated the autophagy pathway, western blot analysis was used to evaluate the expression levels of the autophagy-related proteins. The results are shown in Fig. 5G and H. As the concentration or the treatment time of SchA increased, the protein expression levels of LC3-II were significantly increased. However, p62 protein expression was increased, suggesting that p62 was not degraded during the autophagic flux. This process was similar to the inhibition of autophagy by chloroquine (CQ) and bafilomycin A1 (BafA1), which inhibited the fusion of autophagosomes and lysosomes (58,59). This observation suggests that whilst SchA did activate autophagy, it hindered the degradation of autophagy substrates, specifically the p62 protein.

Insufficient energy and ATP production is an important cause of autophagy in cells (60). In the present study, an ATP detection kit was used to detect the effects of SchA on intracellular ATP levels. As shown in Fig. 5E and F, as the concentration of SchA increased, the intracellular ATP levels were significantly decreased. This suggests that SchA reduced the levels of intracellular ATP.

AMPK is a key intracellular energy sensor (61). Changes in the protein levels of pAMPKα-T172 and AMPKα in A549 and H1975 cells after SchA treatment were measured using western blot analysis. It was found that with increasing the concentration and the treatment time of SchA, markedly increased AMPKα-T172 phosphorylation in A549 and H1975 cells (Fig. 5G and H). This indicated that SchA activated the AMPK signaling pathway in these cells, consistent with the finding that SchA reduced the intracellular ATP levels. This reduction in intracellular ATP levels and activation of the AMPK signaling pathway may be the cause of autophagy induction by SchA.

Although SchA-treated A549 and H1975 cells exhibited autophagy activation, this process of autophagy did not result in substrate degradation. Commonly used autophagy inhibitors include 3-methyladenine (3-MA), CQ and BafA1 (62). CQ and BafA1 mainly inhibit the fusion of autophagosomes and lysosomes, whereas 3-MA inhibits autophagosome formation (62). In the present study, the autophagosome formation inhibitor, 3-MA was used to inhibit the early stages of autophagy flux and observe the effects of 3-MA on the cytotoxicity of SchA. After the cells were pre-treated with 3-MA for 4 h, 50 µM SchA was added to treat the cells for 24 h, before apoptosis of the cells was measured using flow cytometry. As shown in Fig. 6A and B, the autophagy inhibitor 3-MA did not induce any significant effects on cell apoptosis induced by SchA. This result suggests that SchA-induced autophagy has little impact on apoptosis.